There has been considerable interest in developing nucleic acid based compositions for anti-viral applications, including antisense compositions, double-stranded RNA based compositions, triplex-forming oligonucleotides, ribozymes, etc. Their sequence-specific mode of action holds out the promise of therapeutics having a high level of safety and efficacy. Currently accepted methods of down-regulating viral RNAs of plus strand viruses largely involve directly targeting the plus strand RNAs. For example, antisense oligonucleotides are believed to work by hybridizing to an mRNA, thereby interfering with translation of the mRNA into protein. Antisense oligonucleotides are therefore usually designed to be complementary to a target mRNA. U.S. Pat. No. 6,001,990, for example, “Antisense inhibition of hepatitis C virus”, describes oligonucleotides substantially complementary to sequences of the HCV genomic RNA, i.e., sequences which are complementary to and target the plus strand of the HCV genome. Similarly, it has been thought that RNAi is mechanistically connected to translation so that RNAs that are not translated are refractory to siRNA inhibition, while those that are actively translated are effective targets. See Wang and Carmichael, Microbial. Mol. Biol. Rev. 68: 432-452 (2004) See also, e.g., Yokota at al., EMBO Rep. 4:602-08 (2003), describing siRNA targeting of the 5′ UTR of the HCV genomic RNA. Krönke at al., J. Virol. 78 (7): 3436-46 (2004), evaluated siRNAs directed against HCV genomic RNA including various regions of the coding sequence as well the 5′ NTR, and reported that large sections of the NTRs are resistant to RNAi. They speculated, however, that one sequence directed to the 5′ NTR may actually have targeted the 3′ terminus of the negative strand, possibly contributing to its antiviral activity. Ribozymes appear to be an exception to plus-strand HCV targeting, with U.S. Pat. No. 6,107,028 describing ribozymes targeting the plus and/or negative strands of HCV.
A large number of viruses of clinical relevance produce RNA molecules during replication that are not messenger RNA molecules. For example, positive or plus-strand RNA viruses such as hepatitis C(HCV) generate a so-called negative or minus strand RNA which is complementary to and of opposite polarity (5′ vs. 3′ ends) than the various mRNAs made by the virus. The extreme sequence variability and high rate of mutation of RNA viruses such as HCV provide an impetus to target conserved regions of the viral genomic RNA. However, the complex secondary structure of conserved regions of HCV as well as the presence of cellular and viral proteins binding to these conserved regions in the intracellular environment creates uncertainty as to the applicability of nucleic acid based antiviral approaches to these otherwise preferred target regions. Smith et al. mapped conserved regions of both the plus and minus strands of HCV to determine secondary structure and hybridization accessibility to antisense constructs. See, J. Virol. 76 (19): 9563-74 (2002), “Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences”, also Smith et al., J. Viral Hepat., 11 (2): 115-23 (2004).
Similarly, RNA interference (RNAi) has been used to target the selective destruction of mRNA molecules produced by viruses in strategies aimed at creating effective anti-viral agents. Like antisense, dsRNA-mediated RNAi relies on sequence-specific nucleic acid interactions, but the involvement of the multiprotein RNA-induced silencing complex (RISC) makes it unclear whether antisense accessibility alone correlates to target accessibility for RNAi degradation. Furthermore, since RNAi strategies employ double-stranded RNA molecules (dsRNA), which contain sequences both identical to and complementary to a viral target, a method of targeting e.g. minus-strand RNA in preference to its complementary viral mRNA has not been demonstrated. In turn, the potency of an anti-viral agent that works by selectively targeting e.g. the minus strand (of a plus-strand RNA virus) instead of its mRNA or protein products, has not previously been shown. The present invention provides a method for using RNAi to preferentially target the destruction of e.g. the minus strand of a plus-strand RNA virus, and also provides novel compositions based on this method for potent inhibition of the replication of RNA viruses such as HCV.